ABSTRACT Many large, multi-domain proteins are inherently prone to misfolding and aggregation. As soon as they emerge from the ribosome during translation, they are met by molecular chaperones that help them fold into their native structures. While it is clear that molecular chaperones are essential for correct folding in vivo, we know very little about the underlying mechanisms. The long-term goal of our studies is to define the folding mechanisms of complex, multi-domain proteins in the cell. The aim of this proposal is to determine the function of two nascent chain-binding chaperones, Trigger factor and the DnaK system, in the folding of a model multi-domain protein, EF-G. We will study how these chaperones guide the folding of nascent multi- domain proteins using single-molecule force spectroscopy with optical tweezers. This approach is uniquely suited to manipulate and observe the folding of individual nascent polypeptides in the complex environment of the ribosome and molecular chaperones. We will first measure folding transitions of nascent proteins on the ribosome to define their folding energy landscapes. Then, we will determine how Trigger factor and DnaK change these energy landscapes. Our in vitro experiments will reveal in mechanistic detail and with single- molecule resolution how these chaperones contribute to efficient folding. In the cell, the nascent polypeptide interacts with a network of molecular chaperones and other factors that influence its folding and processing. To complement our single-molecule experiments, we will carry out experiments in live cells to determine folding waypoints of multi-domain proteins in vivo. In addition, we will define how chaperones engage their substrates in living cells. Together, these studies will establish a framework for mechanistically understanding protein folding in vivo. Protein folding is of key importance for cellular protein homeostasis. Protein misfolding and aggregation are a hallmark of many diseases, including neurodegenerative diseases and cancer. The research proposed here may ultimately lead to a better understanding and possible treatments for these diseases.